Interpretive Summary: Seafood-associated illness is a significant problem due to the presence of naturally present Vibrio bacteria. Pandemic strains of Vibrio parahaemolyticus have been particularly problematic in producing high numbers of illnesses, while another Vibrio, Vibrio vulnificus, is associated with a high mortality rate. Together, these vibrios cause the majority of shellfish-associated bacterial illnesses and deaths in the United States. This study was undertaken to determine if genetic factors associated with a pandemic V. parahaemolyticus, known as serotype O3:K6 (Vp O3:K6), influenced the uptake or persistence of Vp O3:K6 in Eastern oysters. Although these genes have been associated with enhanced infectivity and survival of V. parahaemolyticus, they did not influence Vp O3:K6 persistence in seawater or their uptake and persistence in shellfish. In natural seawater and oysters maintained in natural seawater, high levels of Vp O3:K6 declined to negligible levels within 72 h. We determined that the same was true for another strain of pandemic V. parahaemolyticus and for V. vulnificus as well. In sterilized seawater, Vibrio levels increased 1000-fold, indicating that something in the natural seawater was eliminating the vibrios. The cause of these Vibrio reductions was identified as naturally-occurring Vibrio predatory bacteria (VPB), which includes a group of bacteria known as Bdellovibrio-and-like-organisms (BALOs). BALOs and other VPB were isolated in Atlantic, Gulf, and Pacific seawater. When oysters were placed in natural seawater and contaminated with Vp O3:K6, the levels of VPB increased while vibrios declined to negligible levels within 48 h. Vibrios in oysters also decreased to negligible levels as VPB increased in the seawater. Electron microscopic examination confirmed the presence of BALOs and other VPB in seawater. Together, our studies have identified VPB as an important factor in controlling vibrios in their natural setting and suggest that disease outbreaks are more likely to occur when the natural levels of BALOs and other predatory bacteria are suppressed. Ecological studies are needed to identify factors which suppress predatory bacteria in the marine environment.

Technical Abstract:
This study shows that naturally occurring Vibrio predatory bacteria (VPB) exert a major role in controlling pathogenic vibrios in seawater and shellfish. The growth and persistence of Vibrio parahaemolyticus (Vp) and Vibrio vulnificus (Vv) were assessed in natural seawater and in the Eastern oyster, Crassostrea virginica. Pathogens examined were Vv strain VV1003, Vp O1:KUT, Vp O3:K6 and corresponding O3:K6 mutants deficient in the toxRS virulence regulatory gene or the rpoS alternative stress-response sigma factor gene. Vibrios were selected for streptomycin resistance, which facilitated their accurate enumeration. In natural seawater, oysters bioconcentrated each Vibrio strain for 24 h; however, counts rapidly declined to near negligible levels by 72 h. In natural seawater with or without oysters, Vibrio levels decreased over 3-logs to negligible levels within 72 h. Neither toxRS nor rpoS had a significant effect on Vibrio levels. In autoclaved seawater, Vp O3:K6 counts increased 1000-fold over 72 h. Failure of the vibrios to persist in natural seawater and oysters led to screening of the water for VPB on lawns of Vp O3:K6 host cells. Many VPB, including Bdellovibrio-and-like-organisms (Bdellovibrio bacteriovorus, Bacteriovorax stolpii) and Micavibrio aeruginosavorus-like predators, were detected by plaque assay and electron microscopic analysis of plaque-purified isolates from Atlantic, Gulf Coast and Hawaiian seawater. When Vp O3:K6 were added to natural seawater containing trace amounts of VPB, Vibrio counts diminished 3-logs to non-detectable levels while VPB increased by 3-logs within 48 h. We propose a new paradigm that VPB are important modulators of pathogenic vibrios in seawater and oysters.